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Article

Novel BODIPY Conjugates with Myrtenol: Design, Spectral Characteristics, and Possibilities for Practical Application

1
G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, 153045 Ivanovo, Russia
2
Faculty of Medicine and Biology, Kazan State Medical University, 420012 Kazan, Russia
3
Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
4
Institute of Petroleum, Chemistry and Nanotechnologies, Kazan National Research Technological University, 420015 Kazan, Russia
5
Scientific Research Institute of Epidemiology and Microbiology, 420015 Kazan, Russia
6
Institute of Chemistry, Federal Research Center “Komi Scientific Centre”, Ural Branch, Russian Academy of Sciences, 167000 Syktyvkar, Russia
7
Institute of Physics, Kazan Federal University, 420008 Kazan, Russia
*
Author to whom correspondence should be addressed.
Inorganics 2023, 11(6), 241; https://doi.org/10.3390/inorganics11060241
Submission received: 21 April 2023 / Revised: 19 May 2023 / Accepted: 31 May 2023 / Published: 3 June 2023

Abstract

:
The synthesis of new fluorescent probes, based on biocompatible luminophors and exhibiting various specificities, is intensively developed worldwide. Many luminophors contain a hydrophobic group that limits their application for cell staining under vital conditions. Herein, we report the synthesis of two BODIPY molecules—BF2-meso-(4-butan/pentanamido-N-(((1S,5R)-6,6-dimethylbicyclo [3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromides—designed as 10, 11 with a spacer of either four or three CH2 groups in length, respectively. These molecules present conjugates of BODIPY luminophors with (+)-myrtenol via a quaternary ammonium group. Both terpene-BODIPY conjugates demonstrated high fluorescence efficiency in various solvents such as OctOH, DMSO and water, and were characterized by their stability at pH 1.65–9.18. The fusion of the myrtenol, a monocyclic terpene, to the BODIPY fluorophore in the meso-substituent facilitated their penetration into the filamentous fungi Fusarium solani, while impairing the binding of the latter with S. aureus, K. pneumoniae and P. aeruginosa. The additional quaternary ammonium group between the myrtenol and fluorophore moieties restored the bacterial cell-staining while it did not affect the staining of fungi. Finally, the BODIPY conjugate 11 was able to stain both Gram-positive and Gram-negative bacteria by its interaction with their cell wall (or the membrane), as well as penetrating into filamentous fungi F. solani and staining their mitochondria.

1. Introduction

Fluorescent dyes absorbing and emitting in the visible and near-IR regions are promising for the development of fluorescent probes for the labeling and visualization of cells, their organelles and compartments. The dyes absorbing and emitting in the long-wavelength spectra are characterized by a higher efficiency of signal recording due to the higher permeability of the cell [1,2,3,4,5]. Compared to other fluorescent dyes, BODIPY are attractive for practical applications due to their excellent photophysical properties—narrow absorption and emission spectra, intense fluorescence and simple signal modulation [6,7]. In our recent works [8,9] biomarkers 1, 2, based on the BODIPYs with an ester group (-(CH2)nCOOCH3), n = 3 or 4) in dipyrromethene meso-spacer, were described (Figure 1).
The meso-substituted BODIPYs 1, 2 (Figure 1)preferentially stain Gram-positive bacteria and can be proposed for the differential staining of Gram-positive and Gram-negative bacteria in mixed cultures [8]. These luminophores accumulate in the cytoplasm of mammalian cells, and giving a polar micro-speckled staining pattern which is more intensive in tumor cells when compared to normal fibroblasts [8,9]. Moreover, BODIPYs have been proposed as a fluorescent marker when assessing the features and structural changes of cellular membranes of mammalian and fungal cells [9].
One of the promising BODIPY applications in the field of conjugation with biomolecules is devoted to the labeling of vitamins, hormones, lipids and other biomolecules to control the processes of their transport, localization in target cells and metabolism [10,11,12,13]. Being fused with various biomolecules, BODIPY could act as a biomarker of therapeutic agents and solve several tasks, such as depicting the cellular localization of the latter and real-time imaging the suppression of pathogenic microflora and cancer cells, thus providing huge potential for the practical application of BODIPY conjugates in medicine [1].
Recently, we have reported spectral and biological properties of the BODIPY 1 and its conjugate with (+)-myrtenol 3 (BODIPY 4), see Figure 2 [14]. (+)-Myrtenol 3 is a bicyclic monoterpene alcohol found in the essential oils of various plants, whose crude extracts show anti-inflammatory, antinociceptive and antifungal activities, have a pleasant odor, and are widely used in cosmetics [15,16,17,18,19,20]. Moreover, our findings confirmed that (+)-myrtenol is capable of increasing the efficiency of various antimicrobial, antifungal, and antiseptic drugs, exhibiting a pronounced synergy with these compounds [21].
The conjugate 4 exhibits a high quantum yield (to ~100%) in the region of 515−518 nm and effectively penetrates the membranes of both bacterial and fungal cells and, therefore, can be used to examine the features of a broad spectrum of Gram-positive and Gram-negative bacteria, and pathogenic fungi as well [14]. Moreover, BODIPY 4 exhibited a moderate tropism to the subcellular structures in mammalian cells (e.g., mitochondria), thereby providing an attractive scaffold for fluorophores to examine these particular organelles.
BODIPYs 1,2 were used as initial compounds to create conjugates 10, 11 carrying a quaternary ammonium fragments in addition to (+)-myrtenol. The quaternary ammonium compounds, in particular benzyldimethyl[3-(myristoylamino)propyl]ammonium chloride monohydrate, which is commercially available as Miramistin®, exhibit high antibacterial and fungicidal activity [22].
The present paper aims to synthesize BODIPYs 10 and 11 and evaluate their luminescent properties, stability in aqueous and buffer media and affinity to various cells, to discover how the introduction of a charged quaternary ammonium fragments will affect the properties of compounds 10, 11 compared to neutral BODIPY 1, 2, and 4 molecules offered as tools for biological object visualization.

2. Results and Discussion

2.1. Synthesis

BODIPY amides 7, 8 were generated by the alkaline hydrolysis of compounds 1, 2 with alkali in aqueous isopropanol, followed by the reaction of the resulting acids 5, 6 with dimethylaminopropylamine (DMAPA), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), N,N-diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP) in dichloromethane (DCM) (Scheme 1). Furthermore, an excess of myrtenyl bromide 9 was added to BODIPY amides 7 or 8 in DCM at room temperature. After evaporation under low pressure, chromatography was performed to isolate target compounds 10 and 11.

2.2. Spectral Properties

The spectral properties of BODIPY conjugates 10 and 11 were studied in 1-octanol, ethanol, N,N-dimethylformamide, dimethylsulfoxide and pure water. Boron(III) dipyrromethenates 1, 2 and the myrtenol containing BODIPY conjugate 4 served as references. Quantitative spectral characteristics of the studied compounds are shown in Table 1 and Table 2.
Regardless of the meso-substituent modification features, compounds 10, 11 had a two-band electronic absorption spectrum typical of BODIPY dyes 1, 2, 4 (Table 1). The most intense long wavelength S0S1 band were in the range of 495–500 nm, and the shoulder on its left slope appeared at 464–470 nm. In the shorter wavelength region 356–362 nm, a low-intensity broadened S0S2 band occurred. The extinction coefficients (ε) of the intense S0S1 band of compounds 10, 11 were in the range from ~54,000 to ~60,000 l∙mol–1∙cm–1 (Table 1).
Figure 3 shows typical absorption spectra of conjugate 10 in various solvents. Conjugates 10 and 11 and their corresponding BODIPY esters 1, 2, 4 intensely fluoresced at 509–516 nm, depending on the solvent nature (Table 2). The shape of the fluorophores 10 and 11 fluorescence band mirrors the intense S0S1 absorption band with the Stokes shift Δ ν S t = 431–556 сm−1 (Table 2, Figure 3). It should be noted that the introduction of both ester residues (compounds 1, 2, 4) and substituents carrying a charged quaternary ammonium fragment (compounds 10, 11) into the meso-position of the BODIPY core caused a hypsochromic shift (by ~14–18 nm) of the maximum of the intense absorption band and a significant increase in the Stokes shift (up to ~1.4–2.2 fold) compared to meso-unsubstituted tetrametyl-BODIPY, for which Δ ν S t does not exceed 271–461 cm−1 [24]. The observed effect can be caused by the BODIPY chromophore system depolarization because of the total electronic effects of the considered meso-substituents and a slight distortion of the chromophore aromatic system plane [23].
Importantly, in contrast to hydrophobic BODIPYs [23,24,25,26], including dyes 1, 2, and 4, the presence of charged quaternary ammonium fragments in the meso-substituent ensured the solubility of conjugates 10 and 11 to ~10–5 mol·l−1 in water and buffer systems such as tetraoxalate buffer (pH 1.65), phosphate buffer (pH 7.21), and tetraborate buffer (pH 9.18). The luminophores were found to be stable under these environmental conditions (Table 2 and Table 3 and Figure 4) and the intensity of their absorption (a) and fluorescence (b) band spectra almost did not change during 24 h in both acidic and alkaline media (Figure 4), indicating conjugates 10 and 11 as promising for various biomedical applications.
The obtained data (Table 2) fit well with literature data [23] and allow the assumption that the introduction of bulky substituents into the meso-position has a marked effect on the fluorescence efficiency of the BODIPY conjugates 10 and 11. In comparison with BODIPY 1, 2, and 4, for dyes 10 and 11, a significant decrease (up to ~30%) of the fluorescence quantum yield (φ) is apparently driven by a nonradiative energy loss, size increase, conformational rearrangements, and solvation dipole–dipole interactions of the meso-substituent (Table 2). Therefore, for all compounds, the spectral characteristics of fluorophores are almost independent of the solvent nature compared to, for example, phenylmethylene pyridineacetonitrile derivatives [23]. The absorption peak wavelength was barely shifted (by 1–4 nm) in the different solvents, indicating that the ground state charge transfer was hardly affected by the solvent polarity. A slight drop of fluorescence quantum yield of BODIPY 1, 2, and 4 by ~20% was observed upon transfer to more polar media due to the increased solvation of dipole–dipole interactions. On the other hand, for 10 and 11, in both organic (nonpolar or polar) solvents and water, the values of φ almost did not change, while it decreased 1.3–1.8-fold in buffers (Table 3) because of the ion-dipole interactions with buffer components.
The lipophilicity of conjugates 10 and 11 was assessed using the distribution coefficient values of luminophores logP in the two-phase model system 1-octanol–water by the “shake flask” approach at 298.15 K, assuming the two-phase system to be a model cell membrane surrounded by the aqueous medium. A comparative analysis of the obtained and the literature data [23] showed that the partition coefficients increase in the sequence of dyes: 11 (0.14); 10 (0.16); 2 (1.68); 1 (1.83); 4 (1.87). The introduction of a charged quaternary ammonium residue into the meso-position of conjugates 10 and 11 led to a significant increase (almost ~9.3–13.4 fold) in the luminophor’s affinity to hydrophilic media compared to meso-substituted BODIPY esters 1, 2, and 4, probably due to an increase in the hydrophilic solvation interaction efficiency.

2.3. Microscopy

Differential fluorescent staining is a common and widely used tool for both the quantitative and qualitative assessment of pro- and eukaryotic cell sub-population fractions and cellular organelles by using microscopy. Therefore, the development of new dyes with selectivity to given cells or their organelles, i.e., their membranes, cores, mitochondria, etc. is challenging. For fluorophore 2 and its conjugate with terpenoids and quaternary ammonium fragments, its ability to stain bacterial cells and penetrate the matrices of bacterial biofilms was assessed with confocal laser scanning microscopy. S. aureus, P. aeruginosa and K. pneumoniae, three of six most dangerous nosocomial pathogens called ESKAPE and which leadto development of biofilm-associated infections, were chosen as test objects. Mature 48 h biofilms of these bacteria were washed and loaded with PBS containing compounds at a final concentration of 10 μg/mL, incubated for 10 min at room temperature, and CLSM was performed. Additionally, instead of a bright-field view, cells were visualized by staining with the fluorescent dye DAPI.
The pure fluorophore 2 was able to stain the cells of all bacteria, including those embedded into the microbial biofilm matrix, and provided a high level of fluorescence (Figure 5A–C). In this case, the maximal signal was observed for S. aureus (3000 photons), while for Gram-negative P. aeruginosa and K. pneumoniae, the fluorescence intensity was two-fold less (~1500 photons). The fusion of a myrtenol molecule to 2 drastically reduced the staining efficiency. Thus, while the observed fluorescence intensity of stained S. aureus and P. aeruginosa cells decreased 5-fold (compare Figure 5A,E, and Figure 5C,F), almost no staining of K. pneumonia was observed (Figure 5E), also confirmed by the predominantly blue color of the cells in the overlayered image (Figure 6E). In turn, the introduction of an additional quaternary ammonium group into 4 facilitated the interaction of 11 with cells, since only a 1.5–2-fold decrease in fluorescence was observed compared to the pure fluorophore 2 (Figure 5G–I). Notably, 11 was almost completely localized in the membrane/cell wall of S. aureus (Figure 5G) and K. pneumonia (Figure 5H), while P. aeruginosa cells were completely stained (Figure 5I).
In marked contrast to bacteria, 2 was unable to penetrate into F.solani filaments (see Figure 7A,C), while the fusion of BODIPY with either myrtenol or myrtenol and a quaternary ammonium moiety facilitated the penetration of the dye into a fungal cell (see Figure 7D,G). Of note, the 4 and 11 seem to bind with organelles, probably mitochondria, since the treated filaments gave a grainy pattern on images.

3. Materials and Methods

3.1. General

Isopropanol, toluene, and dichloromethane were reagent grade and used without purification. Dimethylaminopropylamine, hexafluorophosphate azabenzotriazole tetramethyl uronium, N,N-diisopropylethylamine, 4-dimethylaminopyridine, tetrabromomethane and triphenylphosphine were purchased from Sigma-Aldrich (St. Louis, MO, USA). (+)-Myrtenol 3 and (+)-myrtenyl bromide 9 were prepared by a known procedure [27]. Reaction products were purified by flash chromatography on a PuriFlash Column, 15μ C18 HP, 6g (eluent gradient water—methanol).
NMR spectra were recorded on a Bruker AVANCE-II-500 spectrometer with operating frequencies of 500 MHz (for 1H) and 125 MHz (for 13C) in CDCl3 solvent using standard Bruker pulse programs. The numbering of atoms in the description of the NMR spectra differs from the numbering in the names of compounds. The spectra are shown in Supplementary Materials (Figures S1–S4).
HRMS mass spectra were obtained on a quadrupole time-of-flight (t, UHR-TOF) Brucker Daltonik GmbH, (Germany, Bremen), maXis impact mass spectrometer using ion Booster source (nebulizer gas nitrogen, a positive ionization polarity, capillary voltage 4000 V). Recording of the spectra was performed in ‘‘TOF MS” mode with a collision energy 10 eV, transfer time 110 µs and with resolution of more than 30,000 full-width half-maximum. Samples with the analyte concentration 5 µmol/L were prepared by dissolving the test compounds in a mixture of methanol (HPLC-UV Grade, LabScan) and water (LC-MS Grade, Panreac) in a 1:1 ratio.

3.2. General Procedure for Synthesis of BODIPYs 7, 8

BODIPY 1 (0.057 mmol) or 2 (0.059 mmol) in isopropanol (5 mL) were stirred with 0.1 N NaOH (1 mL) at room temperature for 1 h and evaporated. Then, toluene (10 mL) and diluted aqueous HCl were added to the mixtures with intensive stirring for neutralization. The organic phases were separated and evaporated under low pressure. To round bottom flasks containing not-isolated 5 or 6 in DCM (15 mL) HATU (0.060 mmol), DIPEA (0.115 mmol), DMAP (0.060 mmol) and equimolar amounts of DMAPA were added. Reaction mixtures were stirred for 1 h at room temperature. After evaporation under low pressure, reaction products were purified to afford amides 7 (orange crystals, yield 70%) and 8 (orange crystals, yield 74%).

3.3. General Procedure for Synthesis of BODIPY Conjugates 10, 11

To BODIPY amides 7 (0.039 mmol) or 8 (0.044 mmol) in round bottom flasks, DCM (10 mL) and an excess of myrtenyl bromide 9 (0.13 mmol) were added and stirred for 40 min at room temperature. After evaporation under low pressure, chromatography was performed to provide target compounds 10 and 11.
BF2-meso-(4-pentanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromide (10). Viscous orange oil. Yield: 70 %. [α]23D = +8.0° (c 0.16; MeOH).
NMR 1H (CDCl3) δ, ppm: 0.74 (s, 6H, CH3-1,2), 1.06 (s, 6H, CH3-21,22), 1.24 (s, 2H, CH2-17), 1.56 (m, 4H, CH2-5,16), 1.77 (m, 2H, CH2-6), 1.91 (s, 2H, CH2-5′), 2.00 (s, 5H, CH-4, CH2-15), 2.10 (s, 2H, CH2-6′), 2.14 (m, 2H, CH2-18), 2.34 (s, 1H, CH-3), 2.37 (s, 6H, CH3-19,24), 2.86 (s, 6H, CH3-9,10), 2.90 (m, 2H, CH2-11), 3.26 (s, 1H, CH-20,23), 3.62 (m, 4H, CH2-8,12), 3.74 (q, 2H, CH2-13, J = 104.3;11.9), 5.98 (s, 1H, CH-7), 8.19 (s, 1H, NH-14).
NMR 13C {1H} (CDCl3) δ, ppm: 14.6 (CH3-21,24), 16.7 (CH3-22), 21.4 (CH3-19), 23.0 (CH2-12), 26.1 (CH3-1,2), 26.3 (CH2-17), 28.5 (CH2-16), 31.6 (CH2-18), 32.0 (CH2-6), 32.3 (CH2-5), 35.8 (CH2-15), 36.1 (C-25), 38.2 (CH2-13), 39.8 (CH-4), 47.2 (CH-3), 49.9 (CH3-10), 50.1 (CH3-9), 63.4 (CH2-11), 70.1 (CH2-8), 121.9 (CH-20, C-32), 125.5 (C-31), 128.4 (CH-7), 129.2 (C-26), 131.5 (CH-23), 135.9 (C-27), 136.8 (C-29), 141.1 (C-33), 146.6 (C-28), 153.9 (C-30), 173.9 (CO-14).
HRMS: m/z [M]+ calcul. for C33H50BF2N4O+: 567.60; found: 567.4046.
BF2-meso-(4-butanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromide (11). Viscous orange oil. Yield: 61%. [α]23D = +35.5° (c 0.14; MeOH).
NMR 1H (CDCl3) δ, ppm: 0.85 (s, 6H, CH3-1,2), 1.17 (s, 6H, CH3-21,22), 1.33 (s, 2H, CH2-17), 1.43 (m, 2H, CH2-16), 1.96 (m, 2H, CH2-5), 2.09 (s, 2H, CH2-6), 2.20 (s, 2H, CH2-5′), 2.26 (s, 1H, CH-4), 2.43 (s, 2H, CH2-6′), 2.47 (s, 6H, CH3-19,24), 2.57 (m, 2H, CH2-15), 2.63 (s, 1H, CH-3), 3.05 (m, 2H, CH2-11), 3.09 (s, 6H, CH3-9,10), 3.39 (s, 2H, CH-20,23), 3.79 (q, 2H, CH2-12, J = 104.3;11.9), 3.84 (m, 2H, CH2-8), 3.89 (q, 2H, CH2-13, J = 104.3;11.9), 6.10 (s, 1H, CH-7), 8.62 (s, 1H, NH-14).
NMR 13C {1H} (CDCl3) δ, ppm: 14.4 (CH3-21,24), 16.6 (CH3-22), 21.3 (CH2-12, CH3-19), 22.7 (CH3-1,2), 25.9 (CH2-17), 27.9 (CH2-16), 31.9 (CH2-6), 32.2 (CH2-5), 36.2 (CH2-15), 38.1 (C-25), 39.7 (CH2-13), 45.9 (CH-4), 47.2 (CH-3), 50.0 (CH3-9,10), 63.2 (CH2-11), 70.2 (CH2-8), 121.6 (CH-20, C-32), 128.2 (C-31), 129.8 (CH-7), 130.9 (C-26), 131.5 (CH-23), 135.8 (C-27), 136.7 (C-29), 141.0 (C-33), 146.0 (C-28), 153.8 (C-30), 173.3 (CO-14).
HRMS: m/z [M]+ calcul. for C32H48BF2N4O+: 553.57; found: 553.3889.

3.4. Instruments and Methods

Fluorescence quantum yields (φ) were determined in accordance with the equation [28] according to the equation:
φ = φ s t · S x S s t · A s t A x · ( n x n s t ) 2
where φ and φst are the quantum yields of the sample and standard, Sx and Sst are the area under the emission spectrum of the sample and the standard, Ax and Ast are the optical density in the absorption spectrum of the sample and the standard at the excitation wavelength, and n is the refractive index of the medium. As a standard for determining the fluorescence quantum yield, we used ethanol solution of Rhodamine 6G with a known [29] value of the quantum yield (φ = 0.94) in ethanol.
The compound distribution coefficient (P) was obtained by measuring the dye concentrations in the water–1-octanol phases. BODIPYs were dissolved in 1-octanol (4.2–7.1 μM). The solutions were mixed with water (1 to 1). The mixture was stirred for 8 h at 25 °C. After equilibration, the systems were separated. The logP was calculated by Equation (1): logP = log([dye]1-octanol/[dye]water).

3.5. Strains and Growth Conditions

Staphylococcus aureus subsp. aureus ATCC® 29213™, Pseudomonas aeruginosa АТСС® 27853™ and Klebsiella pneumoniae subsp. pneumoniae АТСС® 13883™ were used in the study. Bacteria were stored as a 50% glycerol stock at −80 °C and maintained in the LB medium. Biofilms were grown for 48 h in the BM broth (glucose 5 g, peptone 7 g, MgSO4 × 7H2O 2.0 g and CaCl2 × 2H2O 0.05 g in 1.0 L tap water) [30,31,32] at 37 °C under static conditions in imaging cover slips (Eppendorf). Furthermore, a clinical isolate of Fusarium solani F-417 isolated from the skin was obtained from the collection of the Kazan Institute of Microbiology and Epidemiology (Kazan, Russia). A 5-day-old culture of fungi grown on Sabouraud’s solid nutrient medium was suspended in RPMI (Roswell Park Memorial Institute) broth and seeded with fungi until 1 × 105cells/mL.

3.6. Microscopy

The penetration of compounds into mature bacterial biofilms and fungal cells was assessed with confocal laser scanning microscopy. For this, mature 48 h biofilms on cell imaging cover slips (Eppendorf) were stained with DAPI (5 μg/mL) and compounds (10 μg/mL). Fungal cells were stained with compounds (10 μg/mL) for 10 min. To assess the localization of the tested fluorophores in fungal cells, a fluorescent blue dye, calcofluor white (CFW), was used. Compound localization was analyzed by an Olympus IX83 inverted microscope supplemented with a STEDYCON ultrawide extension platform.

4. Conclusions

Thus, we demonstrated the effect of various meso-substituents in BODIPY molecules (BF2-meso-(4-butan/pentanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromides) on their properties. Thus, the fusion of myrtenol, a monocyclic terpene, to the BODIPY fluorophore in the meso-substituent facilitated its penetration into filamentous fungi F. solani, while impairing the binding of the latter with S. aureus, K. pneumoniae and P. aeruginosa. The additional quaternary ammonium group between myrtenol and the fluorophore moieties restored the bacterial cells staining, but did not affect staining of the fungi. Both the BODYPI fusions with solely myrtenol, and the myrtenol bound with quaternary ammonium, did not bind with the fungal cell wall and gave grainy images, apparently because of binding with organelles, probably mitochondria. The BODIPY fused to myrtenol via the quaternary ammonium group exhibited high fluorescence efficiency in media of various natures and stabilities over a wide pH range, demonstrating the practical potential of the synthesized BODIPY conjugates as fluorescent markers for biomedical applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/inorganics11060241/s1, Figure S1: 1H NMR spectrum of BF2-meso-(4-pentanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromide (10) in CDCl3, Figure S2: 13C NMR spectrum of BF2-meso-(4-pentanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromide (10) in CDCl3, Figure S3: 1H NMR spectrum of BF2-meso-(4-butanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromide (11) in CDCl3, Figure S4: 13C NMR spectrum of BF2-meso-(4-butanamido-N-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-N,N-dimethylpropan-1-aminium)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene bromide (11) in CDCl3.

Author Contributions

Conceptualization, L.E.N. and R.S.P.; formal analysis, E.V.A., M.B.B., I.R.G., S.A.L., L.L.F., O.V.O., I.Z.R. and E.Y.T.; funding acquisition, G.B.G., L.E.N. and A.R.K.; investigation, G.B.G., E.V.A., M.B.B., I.R.G., S.A.L., L.L.F., I.Z.R. and E.Y.T.; methodology, G.B.G., E.V.A., M.B.B., L.E.N., I.R.G., R.S.P., S.A.L., L.L.F., I.Z.R. and E.Y.T.; project administration, G.B.G., L.E.N. and A.R.K.; supervision, R.S.P. and V.V.K.; visualization, G.B.G., E.V.A., L.E.N., I.R.G., R.S.P., S.A.L., I.Z.R. and A.R.K.; writing—original draft, G.B.G., L.E.N., I.R.G., S.A.L., I.Z.R. and A.R.K.; writing—review and editing, G.B.G., L.E.N. and A.R.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was performed with financial support from the Russian Science Foundation (grant N 20-64-47014 for A.R.K. (https://rscf.ru/en/project/20-64-47014/, accessed on 15 April 2023)—microscopy and NMR, grant N 20-63-47026 for E.V.A. (https://rscf.ru/en/project/20-63-47026/, accessed on 15 April 2023)—the spectral investigations and pH-stability) and Kazan State Medical University (Project N 58-012-2022 for L.E.N.—synthetic procedures).

Data Availability Statement

Not applicable.

Acknowledgments

This work was performed in frames of the Strategic Academic Leadership Program (PRIORITY-2030) of Kazan Federal University.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Structural representation of the BODIPYs 1, 2.
Figure 1. Structural representation of the BODIPYs 1, 2.
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Figure 2. Structural representation of (+)-myrtenol 3 and BODIPY conjugate 4.
Figure 2. Structural representation of (+)-myrtenol 3 and BODIPY conjugate 4.
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Scheme 1. Synthesis of compounds 10, 11.
Scheme 1. Synthesis of compounds 10, 11.
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Figure 3. The normalized absorption (a) and fluorescence (b) spectra of conjugate 10 in different solvents.
Figure 3. The normalized absorption (a) and fluorescence (b) spectra of conjugate 10 in different solvents.
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Figure 4. The absorption (a) and fluorescence (λexc = 480 nm) (b) spectra of conjugate 11 (c ~10−6 mol∙L−1) in time at different pH values.
Figure 4. The absorption (a) and fluorescence (λexc = 480 nm) (b) spectra of conjugate 11 (c ~10−6 mol∙L−1) in time at different pH values.
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Figure 5. The penetration of compounds into bacterial cells. Solely fluorophore (2, AC), fluorophore fused with myrtenol (4, DF) and fluorophore fused with myrtenol via quaternary ammonium fragments (11, GI) were added to preformed and PBS-washed 48 h old biofilms of S. aureus (A,D,G), K. pneumoniae (B,E,H) and P. aeruginosa (C,F,I) until final concentrations of 10 µg/mL. After 10 min incubation, cells were analyzed by confocal laser scanning microscopy with excitation/emission at 490/525 nm. Bars correspond to 5 µm in plain view and to 10 µm in Z-stacks. To comparatively evaluate the fluorescence intensity, the maximal photon counts (shown on the right as heat map legend) were normalized by compound molecular weights.
Figure 5. The penetration of compounds into bacterial cells. Solely fluorophore (2, AC), fluorophore fused with myrtenol (4, DF) and fluorophore fused with myrtenol via quaternary ammonium fragments (11, GI) were added to preformed and PBS-washed 48 h old biofilms of S. aureus (A,D,G), K. pneumoniae (B,E,H) and P. aeruginosa (C,F,I) until final concentrations of 10 µg/mL. After 10 min incubation, cells were analyzed by confocal laser scanning microscopy with excitation/emission at 490/525 nm. Bars correspond to 5 µm in plain view and to 10 µm in Z-stacks. To comparatively evaluate the fluorescence intensity, the maximal photon counts (shown on the right as heat map legend) were normalized by compound molecular weights.
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Figure 6. The penetration of compounds into bacterial cells. Solely fluorophore (2, AC), fluorophore fused with myrtenol (4, DF) and fluorophore fused with myrtenol via quaternary ammonium fragments (11, GI) were added to preformed and PBS-washed 48 h old biofilms of S. aureus (A,D,G), K. pneumoniae (B,E,H) and P. aeruginosa (C,F,I) until final concentrations of 10 µg/mL. DAPI was added until a final concentration of 5 µg/mL. After 10 min incubation, images were captured by confocal laser scanning microscope with excitation/emission at 359/457 and 490/525 nm and overlayered. Bars correspond to 5 µm in plain view and to 10 µm in Z-stacks.
Figure 6. The penetration of compounds into bacterial cells. Solely fluorophore (2, AC), fluorophore fused with myrtenol (4, DF) and fluorophore fused with myrtenol via quaternary ammonium fragments (11, GI) were added to preformed and PBS-washed 48 h old biofilms of S. aureus (A,D,G), K. pneumoniae (B,E,H) and P. aeruginosa (C,F,I) until final concentrations of 10 µg/mL. DAPI was added until a final concentration of 5 µg/mL. After 10 min incubation, images were captured by confocal laser scanning microscope with excitation/emission at 359/457 and 490/525 nm and overlayered. Bars correspond to 5 µm in plain view and to 10 µm in Z-stacks.
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Figure 7. The penetration of compounds into filamentous fungi (Fusarium solani). The solely fluorophore (2, AC), fluorophore fused with myrtenol (4, DF) and fluorophore fused with myrtenol via quaternary ammonium fragments (11, GI) were added to a F. solani filament suspension until the final concentrations of 10 µg/mL. Calcofluor (CFW) white was added until a final concentration of 5 µg/mL. After 10 min, incubation images were captured by confocal laser scanning microscope with excitation/emission at 359/457 and 490/525 nm and overlayered. Bars correspond to 50 µm.
Figure 7. The penetration of compounds into filamentous fungi (Fusarium solani). The solely fluorophore (2, AC), fluorophore fused with myrtenol (4, DF) and fluorophore fused with myrtenol via quaternary ammonium fragments (11, GI) were added to a F. solani filament suspension until the final concentrations of 10 µg/mL. Calcofluor (CFW) white was added until a final concentration of 5 µg/mL. After 10 min, incubation images were captured by confocal laser scanning microscope with excitation/emission at 359/457 and 490/525 nm and overlayered. Bars correspond to 50 µm.
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Table 1. Characteristics of electronic absorption spectra of compounds 1, 2, 4, 10, and 11 in various solvents.
Table 1. Characteristics of electronic absorption spectra of compounds 1, 2, 4, 10, and 11 in various solvents.
SolventCompound
λ max a b s , nm (ε, l·mol−1·сm−1)
1 [23]2 [23]4 [23]1011
1-OсtOH502 (61,467)
358–364
499 (72,443)
357–364
501 (56,234)
357–364
500 (60,583)
358–364
499 (59,302)
360–365
EtOH498 (60,491)
358–360
497 (72,444)
356–361
498 (45,708)
355–361
499 (57,420)
357–362
498 (58,379)
356–362
DMF497 (61,659)
358–362
497 (76,709)
357–363
499 (48,669)
359–369
498 (56,846)
356–361
498 (55,615)
358–363
DMSO498 (56,469)
359–362
497 (66,069)
356–363
499 (47,657)
355–360
500 (60,108)
357–360
498 (54,866)
357–361
Water497 (56,853)
358–360
495 (54,748)
357–362
Notes: λ max a b s —maximum absorption bands (S0S1, S0S2), nm; ε—molar extinction coefficient, l·mol−1·cm−1.
Table 2. The luminescent characteristics of compounds 1, 2, 4, 10, and 11 in various solvents.
Table 2. The luminescent characteristics of compounds 1, 2, 4, 10, and 11 in various solvents.
SolventCompound
1 [23]2 [23]4 [23]1011
λ max f l
( λ e x 470)
nm
Δ ν S t
cm−1
φ λ max f l
( λ e x 470) nm
Δ ν S t
cm−1
φ λ max f l
( λ e x 470) nm
Δ ν S t
cm−1
φ λ max f l
( λ e x 480) nm
Δ ν S t
cm−1
φ λ max f l
( λ e x 480) nm
Δ ν S t
cm−1
φ
1-OctOH5165400.965156230.985165800.955114310.705114710.66
EtOH5135870.895115510.875125490.855114710.755104730.70
DMF5157030.795125900.855156220.825115110.745104730.69
DMSO5156620.745125900.785156220.785115110.745104730.72
Water5105130.725095560.67
Notes: λex, λflmax—excitation and emission maxima, respectively, nm; ∆νSt—Stokes shift, cm−1; φ—fluorescence quantum yield.
Table 3. The fluorescence quantum yields of dyes 10 and 11 in different buffer systems.
Table 3. The fluorescence quantum yields of dyes 10 and 11 in different buffer systems.
CompoundBuffer Solution
Potassium Tetraoxalate Buffer, pH 1.65Phosphate
Buffer,
pH 7.21
Sodium Borate
Buffer
pH 9.18
φ
100.410.460.46
110.460.490.47
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Guseva, G.B.; Antina, E.V.; Berezin, M.B.; Nikitina, L.E.; Gilfanov, I.R.; Pavelyev, R.S.; Lisovskaya, S.A.; Frolova, L.L.; Ostolopovskaya, O.V.; Rakhmatullin, I.Z.; et al. Novel BODIPY Conjugates with Myrtenol: Design, Spectral Characteristics, and Possibilities for Practical Application. Inorganics 2023, 11, 241. https://doi.org/10.3390/inorganics11060241

AMA Style

Guseva GB, Antina EV, Berezin MB, Nikitina LE, Gilfanov IR, Pavelyev RS, Lisovskaya SA, Frolova LL, Ostolopovskaya OV, Rakhmatullin IZ, et al. Novel BODIPY Conjugates with Myrtenol: Design, Spectral Characteristics, and Possibilities for Practical Application. Inorganics. 2023; 11(6):241. https://doi.org/10.3390/inorganics11060241

Chicago/Turabian Style

Guseva, Galina B., Elena V. Antina, Mikhail B. Berezin, Liliya E. Nikitina, Ilmir R. Gilfanov, Roman S. Pavelyev, Svetlana A. Lisovskaya, Larisa L. Frolova, Olga V. Ostolopovskaya, Ilfat Z. Rakhmatullin, and et al. 2023. "Novel BODIPY Conjugates with Myrtenol: Design, Spectral Characteristics, and Possibilities for Practical Application" Inorganics 11, no. 6: 241. https://doi.org/10.3390/inorganics11060241

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